Clot Contraction Drives the Translocation of Procoagulant Platelets to Thrombus Surface.

Objective- After activation at the site of vascular injury, platelets differentiate into 2 subpopulations, exhibiting either proaggregatory or procoagulant phenotype. Although the functional role of proaggregatory platelets is well established, the physiological significance of procoagulant platelets, the dynamics of their formation, and spatial distribution in thrombus remain elusive. Approach and Results- Using transmission electron microscopy and fluorescence microscopy of arterial thrombi formed in vivo after ferric chloride-induced injury of carotid artery or mechanical injury of abdominal aorta in mice, we demonstrate that procoagulant platelets are located at the periphery of the formed thrombi. Real-time cell tracking during thrombus formation ex vivo revealed that procoagulant platelets originate from different locations within the thrombus and subsequently translocate towards its periphery. Such redistribution of procoagulant platelets was followed by generation of fibrin at thrombus surface. Using in silico model, we show that the outward translocation of procoagulant platelets can be driven by the contraction of the forming thrombi, which mechanically expels these nonaggregating cells to thrombus periphery. In line with the suggested mechanism, procoagulant platelets failed to translocate and remained inside the thrombi formed ex vivo in blood derived from nonmuscle myosin ( MYH9)-deficient mice. Ring-like distribution of procoagulant platelets and fibrin around the thrombus observed with blood of humans and wild-type mice was not present in thrombi of MYH9-knockout mice, confirming a major role of thrombus contraction in this phenomenon. Conclusions- Contraction of arterial thrombus is responsible for the mechanical extrusion of procoagulant platelets to its periphery, leading to heterogeneous structure of thrombus exterior.

Calpain-controlled detachment of major glycoproteins from the cytoskeleton regulates adhesive properties of activated phosphatidylserine-positive platelets.

In resting platelets, adhesive membrane glycoproteins are attached to the cytoskeleton. On strong activation, phosphatidylserine(PS)-positive and -negative platelet subpopulations are formed. Platelet activation is accompanied by cytoskeletal rearrangement, although the glycoprotein attachment status in these two subpopulations is not clear. We developed a new, flow cytometry-based, single-cell approach to investigate attachment of membrane glycoproteins to the cytoskeleton in cell subpopulations. In PS-negative platelets, adhesive glycoproteins integrin αIIbß3, glycoprotein Ib and, as shown for the first time, P-selectin were associated with the cytoskeleton. In contrast, this attachment was disrupted in PS-positive platelets; it was retained to some extent only in the small convex regions or 'caps'. It correlated with the degradation of talin and filamin observed only in PS-positive platelets. Calpain inhibitors essentially prevented the disruption of membrane glycoprotein attachment in PS-positive platelets, as well as talin and filamin degradation. With the suggestion that detachment of glycoproteins from the cytoskeleton may affect platelet adhesive properties, we investigated the ability of PS-positive platelets to resist shear-induced breakaway from the immobilized fibrinogen. Shear rates of 500/s caused PS-positive platelet breakaway, but their adhesion stability increased more than 10-fold after pretreatment of the platelets with calpain inhibitor. In contrast, the ability of PS-positive platelets to adhere to immobilized von Willebrand's factor at 100/s was low, but this was not affected by the preincubation of platelets with a calpain inhibitor. Our data suggest that calpain-controlled detachment of membrane glycoproteins is a new mechanism that is responsible for the loss of ability of the procoagulant platelets to resist detachment from thrombi by high shear stress.

Procoagulant platelets form an α-granule protein-covered "cap" on their surface that promotes their attachment to aggregates.

Strongly activated "coated" platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbß3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein "coat." Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as "cap," a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbß3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather "capped") platelets to become incorporated into thrombi despite their lack of active integrins.

Two types of procoagulant platelets are formed upon physiological activation and are controlled by integrin α(IIb)ß(3).

OBJECTIVE: Phosphatidylserine (PS) externalization by platelets upon activation is a key event in hemostasis and thrombosis. It is currently believed that strong stimulation of platelets forms 2 subpopulations, only 1 of which expresses PS. METHODS AND RESULTS: Here, we demonstrate that physiological stimulation leads to the formation of not 1 but 2 types of PS-expressing activated platelets, with dramatically different properties. One subpopulation sustained increased calcium level after activation, whereas another returned to the basal low-calcium state. High-calcium PS-positive platelets had smaller size, high surface density of fibrin(ogen), no active integrin α(IIb)ß(3), depolarized mitochondrial membranes, gradually lost cytoplasmic membrane integrity, and were poorly aggregated. In contrast, the low-calcium PS-positive platelets had normal size, retained mitochondrial membrane potential and cytoplasmic membrane integrity, and combined retention of fibrin(ogen) with active α(IIb)ß(3) and high proaggregatory function. Formation of low-calcium PS-positive platelets was promoted by platelet concentration increase or shaking and was decreased by integrin α(IIb)ß(3) antagonists, platelet dilution, or in platelets from kindlin-3-deficient and Glanzmann thrombasthenia patients. CONCLUSIONS: Identification of a novel PS-expressing platelet subpopulation with low calcium regulated by integrin α(IIb)ß(3) can be important for understanding the mechanisms of PS exposure and thrombus formation.

Identification of different proaggregatory abilities of activated platelet subpopulations.

Blood platelets are anucleate cell fragments that play a critically important role in hemostasis and thrombosis. Platelets are activated with various agonists that allow them to aggregate, thus forming either hemostatic plugs or pathologic thrombi. Recent studies have revealed that at least two activated platelet subpopulations are formed upon potent stimulation of platelets with collagen and/or thrombin. One of these subpopulations consists of so-called coated platelets that express high levels of phosphatidylserine and retain α-granule proteins, including fibrinogen, on their surface. They also have reduced levels of the main aggregation receptor-activated glycoprotein IIb-IIIa, which might indicate a defect in their proaggregatory ability. In this study, the proaggregatory abilities of coated and noncoated platelets were assessed by means of light transmission aggregometry of suspensions with varying ratios of platelets from one subpopulation to those of a different subpopulation. A mathematical model of platelet aggregation in heterogeneous mixtures was developed to assist in the analysis of experimental data. Flow cytometry was employed to monitor platelet recruitment into aggregates and the ability of platelets to bind external fibrinogen. Finally, confocal microscopy was used to image coated platelets involved into aggregates formed by mechanical shaking. The obtained data revealed to our knowledge a novel mechanism regulating aggregate formation of platelet subpopulations: coated platelets cannot aggregate with each other but can be recruited into aggregates by noncoated platelets.